scFv Conversion into scFv-Fc

Conversion of scFv into scFv-Fc Fusions

A rapid method to validate phage clones.

A

fter biopanning and identification of lead clones, validation must be completed with free antibodies. The use of an amber codon between the scFv and the pIII minor coat protein to switch to soluble expression too often yields poor expression levels and requires extra purification steps. Moreover, scFvs are monovalent entities often binding significantly less than the equivalent Fab fragment or the antibodies they are deriving from.

TGEX-SCblue is a mammalian expression vector especially designed for the conversion of scFv phage clones into scFv-Fc fusion proteins that can be quickly expressed by transient expression into HEK293 cells with yields between 10 mg/l and 100 mg/l or more in just a few days. The bivalent Fc fusions are secreted in the supernatant and in most cases will reproduce the binding of a fully reconstructed mAb. Transient transfections can be done easily in 24-wells and 48-wells plates in parallel.

The pelbK leader sequence is a hybrid between a human Kappa leader and the bacterial pelB leader; this leader drives efficient secretion of antibodies in the supernatant (1, 2). A common SfiI site found in all pADL phagemids and the pelBK leader enables seamless transfer of scFv or VHH domains between the two systems (Fig. 1).

Figure 1. Conversion of scFv into scFv-Fc fusion. TGEX-SCblue vector contains a double SfiI restriction site found in all pADL phagemids. A lacZ fragment in the cloning site allows for quick identification of recombinants by blue/white selection.

Advantages of transient mammalian expression for antibody validation

  • Improved binding.

  • Easy detection.

  • High yields.

  • No purification.

References

  1. Valadon, P., Garnett, J.D., Testa, J.E., Bauerle, M., Oh, P. and Schnitzer, J.E. (2006). Proc Natl Acad Sci U S A. 103(2):407-12.

  2. Yoon, H., Song, J. M., Ryu, C. J., Kim, Y.-G., Lee, E. K., Kang, S., & Kim, S. J. (2012). BMC Biotechnology, 12(1), 62.

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